The inositol 1,4, 5 - trisphosphate receptors (IP3Rs) are large integral membrane proteins that function as the intracellular IP3-gated Ca 2+ release channels and govern rapid fluxes of Ca2+ ions from the endoplasmic reticulum into cytoplasm, thereby, playing a key role in a wide range of physiological functions including neurotransmitter release, fertilization, hormone secretion, gene transcription, metabolic regulation, apoptosis and muscle contraction. Abnormal regulation of Ca2+ homeostasis has been implicated in numerous human diseases such as cardiac hypertrophy, heart failure, hereditary ataxias, osteoporosis, atherosclerosis and some migraines. The long-term objectives of this project are to elucidate the molecular mechanisms of the IP3-induced Ca2+-gating through structure-function analysis of the IP3R channel complex and to define how defects in this channel protein can cause abnormal regulation of cell Ca2+ level underlying human diseases. The proposed project aims to utilize the electron cryomicroscopy and computer reconstruction techniques in conjunction with biochemical, electrophysiological, molecular and computational approaches to delineate the structural domains in the 3-D architecture of the IP3R1 (cerebellar isoform of IP3R) and to define structural steps underlying channel gating. The specific aims of this proposal are: 1) resolve the 3-D structure of the native IP3R1 in open and closed states; 2) ascertain topology of functional domains within quaternary structure of IP3R1; 3) elucidate the effects of calmodulin on the 3-D structure of the channel; 4) determine the 3-D structure of the recombinant constitutively open IP3RI. Proposed structural studies will exploit "single particles" approach, standing for isolated unordered particles. Thus, the purified IP3R1 channel particles will be trapped in different functional states by embedding in a thin layer of vitreous ice in the presence of channel specific modulators and then directly visualized in electron cryomicroscope. Sequence-specific antibodies will be employed to map regions of the primary sequence of the IP3R1, which are predicted to control intrinsic channel properties, in its 3-D structure. We anticipate that results from proposed studies will provide a three-dimensional framework for functional interpretations of the channel gating on which to base future biochemical, electrophysiological, and genetic experiments.